Ornithine transcarbamoylase is one of four urea cycle enzymes. It has been shown that the metal ion Zn2+ induces substrate cooperativity in this enzyme and acts as a specific allosteric cofactor for its regulation in vitro. This facet of enzyme-metal interactions has not been studied previously. Because Zn2+ is not tightly bound to ornithine transcarbamoylase, the finding also underscores one of the problems in our current understanding of metabolism in that many of the allosteric effectors which control the action of futile-cycle enzymes have yet to be recognized. The fundamental objective of this research is to examine at the molecular level the catalytic action and the regulation of ornithine transcarbamoylase. This investigation will focus on the role of Zn2+ in the mechanism of allosteric control and the structural basis of substrate specificity. The following questions will be addressed: how does Zn2+ trigger intersubunit transition, what determines the metal and substrate specificity of the enzyme, and can this mode of allosteric control be a general mechanism hitherto unrecognized for other multimeric enzymes? The specific aims of this proposal are: (1) to characterize the catalytic properties of the enzyme by a detailed kinetic study; (2) to analyze the nature of interactions between the enzyme and its ligands by magnetic resonance and chemical methods; (3) to analyze the structural aspects of binding and allosteric interactions by physical and spectroscopic techniques; (4) to perform a systematic replacement of amino acids in the enzyme by site directed mutagenesis; and (5) to obtain single enzyme crystals. Deficiency in ornithine transcarbamoylase is the most common enzyme defect of the urea cycle. Lack of this enzyme leads to ammonia intoxification and is a major cause of acute metabolic crises in human neonates. The molecular basis of this deficiency is not understood and can be attributed at present only to the production of inactive enzyme forms.